High Cell Density Cultivation of HEK293 Cells Using a 2L Membrane-Stirred Bioreactor

Area of Science:

• Biotechnology and Bioprocessing
• Cell Culture Engineering
• Recombinant Protein Production

Background:

• Perfusion systems offer advantages for continuous animal cell cultivation at high cell density (HCD), enhancing volumetric productivity and product quality.
• Conventional stirred tank bioreactors (STRs) face challenges in HCD cultivations (>50 × 10^6 cells/mL), including limited oxygen supply, CO2 stripping, high viscosity, and foam.
• Addressing these limitations is crucial for process intensification in biopharmaceutical manufacturing.

Purpose of the Study:

• To investigate a novel membrane-stirrer (MemStir) bioreactor for efficient gas transfer and HCD cultivations.
• To evaluate the MemStir system's performance in overcoming aeration and foam formation issues in perfusion cell culture.
• To assess the MemStir system's suitability for producing recombinant proteins, viruses, and viral vectors.

Main Methods:

• A novel MemStir bioreactor utilizing hollow-fiber membranes for bubble-free gas diffusion was designed and tested.
• Performance was benchmarked against shake flasks and conventional STRs in batch mode using HEK-LTV cells.
• HCD cultivations in perfusion mode were conducted using the MemStir system coupled with an alternating tangential flow system.

Main Results:

• The MemStir system achieved volumetric mass transfer coefficient (kLa) values of 10-30 h^-1, ensuring sufficient oxygen supply for HCD.
• Suspension cell growth in the MemStir system was comparable to conventional STRs, reaching a maximum viable cell concentration of 7.6 × 10^6 cells/mL in batch mode.
• In perfusion mode, HEK-LTV cells reached concentrations up to 92 × 10^6 cells/mL with a specific growth rate of 0.022 h^-1, with stable pH control and no foam formation.

Conclusions:

• The MemStir bioreactor effectively overcomes limitations in oxygen supply and foam formation associated with HCD cultivations.
• The system demonstrates robust performance in both batch and perfusion modes, supporting high cell densities for biopharmaceutical production.
• The MemStir system presents a viable and advantageous alternative for process intensification in animal cell culture.